Epigenetic properties responsible for the broad developmental potential of embryonic stem cells (ES cells), hematopoietic stem cells (HSCs), and other types of stem cells are of considerable interest because these cells are advantageous for a number of therapeutic strategies. Stem cells are of equal interest to cancer biologists because of evidence that many human cancers are caused by the aberrant expansion of cells with stem cell-like properties. Recent studies suggest that genes involved in early developmental decisions are poised for activation through their association with bivalent histone modification domains, consisting of both active and repressive epigenetic marks. However, these bivalent domains are not generally associated with typical tissue-specific genes expressed in differentiated cells. Instead, previous studies of the liver-specific Alb1 gene suggested that typical tissue-specific genes are assembled into inaccessible chromatin structures in pluripotent cells and that, during or after gastrulation, pioneer transcription factors initiate a cascade of events that promotes chromatin decondensation and ultimately leads to transcriptional activation. In contrast to this hypothesis, we recently demonstrated that well-characterized enhancers for three tissue-specific genes, Ptcra, Il12b, and Alb1, are selectively marked by unmethylated CpG dinucleotides in pluripotent ES cells. The unmethylated CpGs appear to result from the binding of transcription factors to the enhancers, even though the binding of these factors in ES cells does not lead to nuclease hypersensitivity and does not always promote histone modifications typically associated with active or silent genes. Preliminary functional studies suggest that the enhancer marks we have observed in ES cells may be critical for transcriptional activation of the tissue-specific genes in differentiated cells. In the absence of the enhancer marks, pre-methylated enhancer- promoter-reporter plasmids were resistant to transcriptional activation in differentiated cells, and were unable to establish unmethylated windows at their enhancers. These results lead to the hypothesis that enhancer marks must be established in early development because the more repressive chromatin environment found in differentiated cells is incompatible with transcriptional activation of unmarked genes. To further explore this hypothesis and better understand the establishment and maintenance of the enhancer marks in ES cells, we will continue our studies of the same three model genes used for our preliminary experiments, the thymocyte- specific Ptcra gene, macrophage/dendritic cell-specific Il12b gene, and liver-specific Alb1 gene. A major goal will be to identify the specific DNA elements required for establishment of the enhancer marks, as a first step toward rigorously analyzing the functional significance of the marks. The enhancer marks will also be evaluated in induced pluripotent stem cells (iPS), as a strategy for better evaluating the establishment and significance of the marks during epigenetic reprogramming. PUBLIC HEALTH RELEVANCE: The molecular features of embryonic stem cells, hematopoietic stem cells, and many other types of stem cells are of considerable interest because of the therapeutic potential of stem cell-derived tissues. This study will explore a recently discovered feature of mouse embryonic stem cells that may be critical for their unique properties.